Identification of sex, age and species-specific proteins on the surface of the harpacticoid copepod Tigriopus japonicus

 Behavioral experiments have shown that male copepods of the species Tigriopus japonicus (Nori) can distinguish species, sex, and developmental stage of potential mates using contact chemoreception. Lectin-binding patterns on the body surface of females have indicated that surface-bound glycoproteins may be important signals in mate choice. In the present study, the proteolytic enzyme trypsin was used to cleave surface proteins from females, reducing their attractiveness to males. The protein fragments released were used to make monoclonal antibodies. Three levels of screening were used to identify monoclonal antibodies that recognized proteins involved in mate recognition. One monoclonal antibody bound to the terminal urosome and lateral prosome of CV females, and its binding significantly decreased female attractiveness to males. Western blotting showed that this antibody bound the trypsin-cleaved fragment and proteins of homogenized CV females and virgin adult females, but did not bind proteins of homogenized males, CIII females, or females of T. californicus or T. fulvus. This antibody recognized proteins on the surface of females that may enable males to discriminate conspecifics, sex, and age. It is likely that this molecule has a central role in the evolution of reproductive isolation in this group. Received: 22 July 1999 / Accepted: 21 March 2000     

Cannibalistic behaviour in the harpacticoid copepod Tigriopus fulvus

The results reported here demonstrate the existence of cannibalistic behaviour in laboratory-reared Tigriopus fulvus (Fischer, 1860) females towards first-stage non-related nauplii; mothers, sparing their own offspring, do not share this behaviour. The phenomenon is related to the probability of nauplius/female encounters. It is suggested that mother/offspring recognition is mediated by a chemical compound.     

Salinity tolerance,salinity preference and temperature tolerance in the high-shore harpacticoid copepod Tigriopus brevicornis

Tigriopus brevicornis (O. F. Müller) were collected in 1992 from rock pools close to U.M.B.S. Millport, Isle of Cumbrae, U.K. and acclimated to various combinations of salinity and temperature for at least 1 wk prior to laboratory experiments. Higher salinities of acclimation enhanced tolerance to high salinity stress, while tolerance of low salinities was hardly affected by acclimation salinity. Acclimation to low temperature (10°C) extended the survivable salinity range for T. brevicornis. High-salinity acclimation enhanced the survivable temperature range. Copepods acclimated to 60 survived significantly lower and higher temperatures than did 34-acclimated individuals. At high temperature, 75-acclimated female copepods had the highest median lethal temperature, 38.9°C. Females were significantly more resistant to high temperatures than males. The copepods were seen to have a very low median lethal temperature when frozen into solid ice for 2 h; 50% mortality occurred at-16.9°C in 10°C, 34-acclimated T. brevicornis. Salinity preference experiments demonstrated an ability to discriminate between salinities differing by as little as 3. Copepods acclimated to 34 chose salinities near their acclimation salinity; individuals acclimated to 5 favoured slightly higher salinities, while copepods acclimated to 60 chose rather lower salinities.     

Feeding deterrent and toxicity effects of apo-fucoxanthinoids and phycotoxins on a marine copepod (Tigriopus californicus)

Using the marine harpacticoid copepod Tigriopus californicus, the effects of phytoplankton feeding deterrents and toxins were differentiated and measured. Eight compounds were tested for feeding deterrence and toxicity responses: four apo-fucoxanthinoids (apo-10′-fucoxanthinal, apo-12′-fucoxanthinal, apo-12-fucoxanthinal, and apo-13′-fucoxanthinone) and four well-known phycotoxins (domoic acid, okadaic acid, microcystin-LR, and a mixture of PSP-1 toxins). Since several of these compounds exhibited both feeding deterrence and toxicity, a model was developed to deconvolute the observed toxicity response from the observed feeding deterrence response, and to classify these compounds based on the degree of toxicity and/or feeding deterrence they exhibited towards T. californicus. Microcystin-LR, the PSP-1 toxins, and the four apo-fucoxanthinoids behaved only as feeding deterrents at low concentrations. Okadaic acid exhibited both toxicity and feeding deterrence at low concentrations, with the threshold concentration for feeding deterrence at a lower level than the threshold concentration for toxicity. Domoic acid acted only as a toxin at low concentrations, with all decreases in feeding resulting from the death of the copepod. Received: 4 November 1996 / Accepted: 3 December 1996     

Mating system of the intertidal copepod Tigriopus californicus

Male Tigriopus californicus clasp immature females (copepodid stages II–V) for a period of up to a week prior to the female's terminal molt; upon maturation (stage VI) the female is inseminated and released. While females can mate anytime after their terminal molt, experiments using electrophoretically-detected genetic markers indicate that each mates only once in her lifetime. No evidence of sperm displacement was observed. Hence, male mating behavior can be interpreted as pre-copulatory mate guarding, a strategy employed to assure that a potential mate has not been previously inseminated. Males minimize the time investment required to insemiate a single female successfully by preferentially choosing to clasp more developmentally-advanced females; males clasped to stage III females will release them in order to clasp stage V females if the latter are present. Since males are capable of multiple mating, under most conditions of population sex ratio, this mating system results in low availability of unclasped, developmentally-advanced females; consequently, males must clasp successively younger (i.e. developmentally less-advanced) females in order to obtain a successful insemination.     

Evaluation of the copepod Tigriopus californicus as a bioassay organism for the detection of chemical feeding deterrents produced by marine phytoplankton

Marine phytoplankton have been shown to use chemical feeding deterrents to reduce or inhibit zooplankton grazing. In order to screen phytoplankton species for feeding deterrent production and to isolate and identify feeding deterrent compounds, a new, rapid, and reliable laboratory bioassay was developed. This bioassay used the laboratory-reared harpacticoid copepod Tigriopus californicus and measured inhibition of feeding by measuring the fecal pellet production rate. The bioassay was capable of detecting deterrent compounds: (1) adsorbed onto ground fish food (a normally palatable food); (2) dissolved in a mixture of seawater and live Thalassiosira pseudonana cells (a species of diatom which had no feeding deterrent activity); and (3) present in live cell cultures. Method (2) was recommended for use in bioassay-guided fractionation (isolation of chemical compounds), as it was reliable, rapid, accurate, and easy to perform with large numbers of samples. The total bioassay time was < 48 h, and data collection required only a microscope. Methanolic cell extracts of several phytoplankton species were screened for feeding deterrent activity. Extracts from the diatom Phaeodactylum tricornutum and the dinoflagellate Gonyaulax grindleyi gave feeding deterrent responses, while extracts from the diatom Thalassiosira pseudonana gave no feeding deterrent responses. Live P. tricornutum cells deterred feeding at densities of 6x10⁵ cells ml^-1. This bioassay should provide a valuable tool in screening phytoplankton for feeding deterrent compounds and determining the chemical nature of these compounds.     

Ontogenetic feeding shifts in the meiobenthic harpacticoid copepod Nitocra lacustris

¹⁴C-radiolabelling experiments indicate that adult stages of the salt-marsh harpacticoid copepod Nitocra lacustris (Schmankevitsch) receive a large part of their nutrition through the ingestion and assimilation of certain diatoms. An abundance of empty diatom frustules occurs in the gutpellet contents of field-collected individuals. Naupliar stages do not ingest diatoms in the laboratory, and nauplii from the field do not contain frustules in their gut pellets. Ingestion of diatoms in the laboratory first occurs during the second or third copepodite stage. ³H-radiolabel expeiments and grazing experiments using bacterium-sized beads adhering to the diatoms indicated that both adults and nauplii ingest bacteria adhering to the outer mucus coating of the diatoms (and probably ingest the diatom mucus itself). Adults ingest bacteria (and probably mucus exopolymer) coincidently while ingesting diatoms. The nauplii ingest these components by scraping the outer surface of the diatoms. SEM observations indicate that diatoms are not punctured by the nauplii during feeding. While diatom mucus and associated bacteria play an (as yet unquantified) role in the nutrition of the adults, these components may comprise the bulk of food resources for naupliar stages.     

Inter-sexual conflicts over copulations in the European starling: evidence for the female mate-guarding hypothesis

Recent studies have suggested that conflicts of interests between the sexes may lead to variation in copulation behavior among pairs. We examined differences in the rate and timing of copulation solicitations and copulations among females of different mating status in the facultatively polygynous starling in an attempt to explain why females copulate repeatedly with their mate. All within-pair copulations were female-solicited indicating that females control copulations in the starling. Before egg laying, females solicited copulations at a high rate (usually more than 2 per hour). In contrast to most other species studied so far, females continued to solicit copulations throughout the egg laying period, and also solicited after egg laying (the latest solicitation occurred when the nestlings were 4 days old). Primary females solicited more copulations than monogamous females both during and after the fertile period. Many copulation solicitations of primary females occurred at the nestbox where their male was singing to obtain an extra female. Both primary and secondary females solicited more copulations after the egg-laying period than monogamous females. A large proportion of female copulation solicitations was refused by the male partner: female solicitation resulted in more male refusals in primary females than in monogamous females. Primary females were more likely to be chased aggressively when they solicited a copulation than monogamous females; most aggressive chases occurred when primary females flew towards their male to solicit copulation when he was singing at another nestbox. Overall, our results demonstrate that there is a conflict over copulation between males and females in polygynous pairs. The conflict presumably relates to the cost of sharing male parental investment: females use copulation solicitation behavior to interrupt their singing males apparently in an attempt to prevent them from becoming polygynous. We present the first empirical evidence that female songbirds use copulation solicitation behavior as a form of mate guarding, often in a non-reproductive context. We did not find a positive relationship between copulation rate during the fertile period and the amount of male parental care as is predicted by the paternity confidence hypothesis.     

What is the sex ratio of harpacticoid copepods in the deep sea?

The sex ratios of deep-sea harpacticoids have been thought to be greatly skewed toward females. The representation of males is notably more equitable (approximately 1:2) at three deep-sea sites (San Diego Trough: 32°52.4N, 117°45.5W, 1 050 m, January 1987; Porcupine Seabight: 51°36.85N, 12°57.30W, 1 369 m, August 1984; northwest Atlantic, 40°27N, 62°20W, 4 820 m, July 1982, June 1983). This ratio conceals extensive variation at the species level. For some species, males are unknown. For other species, males are more numerous than females. Sampling bias against males can occur and may explain the more extreme reports of male rareness in the literature.     

Biological effects of surface active agents on marine animals

The biological effects of 5 surface active agents (the anionic ABS, LAS and LES 3 EO and the nonionic NP 10 EO and TAE 10 EO) on marine fishes, crustaceans and bivalves have been tested in, continuous-flow systems. Concentrations from 100 to 0.5 ppm were normally used. Fishes were found to be more susceptible (96 hLC 50 range: 0.8 to 6.5 ppm) than bivalves (96 hLC50 range: 5 to >100 ppm) while crustaceans were considerably more resistant (96 h LC 50 range: 25 to > 100 ppm). Within each of these 3 systematic groups, more active species were found to be more sensitive than less active species. Developmental stages were also more sensitive than adults. The resistance of crustaceans to surfactants decreases immediately after moulting. The most toxic agent for fishes and decapods was the soft anionic LAS, and for bivalves and barnacles the hard nonionic NP 10 EO. Ability to recover normal behaviour after exposure decreases with increasing concentration and time, and ceases earlier in anionic than in nonionic surfactants. The first reaction to the surfactants is increased activity (avoidance reaction of mobile species), followed successively by inactivation, immobilization and death. Nonionic surfactants affect the equilibrium in fishes. Sublethal effects appear as impaired locomotory activity and breathing rate in fishes and crustaceans, impaired byssus activity and shell closure in Mytilus edulis, burrowing in Cardium edule, Astarte montagui and Astarte sulcata. Siphon retraction is affected in Mya arenaria and Cardium edule, as is also the response to food of the Leander spp.     

Feeding deterrence properties of apo-fucoxanthinoids from marine diatoms. II. Physiology of production of apo-fucoxanthinoids by the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana,and their feeding deterrent effects on the copepod Tigriopus californicus

The marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have been shown to produce apo-fucoxanthinoid compounds which act as feeding deterrents against the harpacticoid copepod Tigriopus californicus. The amounts and types of apo-fucoxanthinoids produced were species specific. Th. pseudonana produced small quantities of apo-12-fucoxanthinal and apo-13-fucoxanthinone only during senescence, while P. tricornutum produced much greater quantities of these two compounds during both log and senescence phases, in addition to producing a third compound, apo-10-fucoxanthinal, only during senescence. For both species, production of apo-fucoxanthinoids increased as the cells entered senescence phase due to phosphate limitation. The amounts of apo-fucoxanthinoids necessary to reduce feeding in T. californicus by 50% ranged from 2.22 to 20.2 ppm. This range was approximately 1000 times lower than the total apo-fucoxanthinoid concentration in P. tricornutum. The amounts of apo-fucoxanthinoids necessary to cause a 50% mortality in a population of T. californicus ranged from 36.8 to 76.7 ppm. Thus, these compounds are present in concentrations which may have ecological significance in the control of bloom formation and grazing. The production of apo-fucoxanthinoids may be a phenomenon common to many diatoms, particularly as they enter senescence due to nutrient limitation.     

Maintenance of a diverse harpacticoid copepod community in microcosm culture

The design and construction of a closed system circulating sea-water tidal estuarine sediment microcosm is described. The microcosm was used to culture mixed species populations of the Harpacticoida (Crustacea: Copepoda) and the evolution of the community and the successional changes are detailed. The communities that developed in each of four replicate microcosms were statistically indistinguishable with respect to the species richness, the numbers of individuals recorded, the log series diversity and the dominance diversity statistics. The most abundant species were the same in each microcosm with the same rank. A diverse community was maintained that did not tend to monoculture; however, the successional changes observed were not apparent in the estuarine communities from which the culture sediments was collected. Rather than a functional reorganisation of the community effecting this, gross environmental changes resulting from a decreased depth of sediment matrix are implicated. The success in maintaining a diverse community is attributed to a provision of a number of nutrient sources.     

An electron-microscope investigation of the cells lining the outer surface of the mantle in some marine molluscs

The ultrastructural details of the cells lining the outer surface of the mantle in Mytilus edulis (L), Cardium edule (L), and Nucula sulcata Bronn. are described. Along the length of the outer mantle fold and the general outer surface of the mantle, the epithelial cells differ, with a progressive reduction in the protein synthetic apparatus and an increase in glycogen, while large numbers of mitochondria are evident in the apical and basal regions.     

Sexual cohabitation as mate-guarding in the leaf-curling spider Phonognatha graeffei Keyserling (Araneoidea, Araneae)

The leaf-curling spider Phonognatha graeffei incorporates a twisted leaf into the central hub of its orb-web that is used as a retreat. This species is unusual among orb-weaving spiders because males cohabit in the leaf retreat with both immature and mature females, mating with the former shortly after the female molts. Cohabitation appears to be a form of mate-guarding because cohabiting males respond agonistically to rival males that venture onto the web, and their behaviour depends upon the reproductive status of the female; males defending immature females are more aggressive than those defending virgin, adult females. Males copulate with previously mated females for significantly longer than with virgin females. Females may cannibalise cohabiting males, which occurs independently of whether the female has been deprived of food. Females that cannibalise a single male do not have a higher fecundity than non-cannibalistic females. Received: 2 February 1996 / Accepted after revision: 27 October 1996     

Mate choice in the face of both inbreeding and outbreeding depression in the intertidal copepod Tigriopus californicus

 In species vulnerable to both inbreeding and outbreeding depression, individuals might be expected to choose mates at intermediate levels of genetic relatedness. Previous work on the intertidal copepod Tigriopus californicus has repeatedly shown that crosses between populations result in either no effect or hybrid vigor in the first generation, and hybrid breakdown in the second generation. Previous work also shows that mating between full siblings results in inbreeding depression. The present study again found inbreeding depression, with full sibling mating causing significant fitness declines in two of the three populations assayed. In the mate choice assays, a single female was combined with two males. Despite the costs of both inbreeding and outbreeding, mate choice showed clear inbreeding avoidance but no clear pattern of outbreeding avoidance. This lack of outbreeding avoidance may be attributed either to the temporary increase in fitness in the F₁ generation or to the absence of selection for premating isolation in wholly allopatric populations with infrequent migration. If this inability to avoid unwise matings is common to other taxa, it may contribute to the problem of outbreeding depression when allopatric populations are mixed together. Received: 18 May 1999 / Accepted: 25 January 2000     

Role of glutamine synthetase in ammonia assimilation by symbiotic marine dinoflagellates (zooxanthellae)

The dinoflagellate symbionts (zooxanthellae) present in many reef corals aid in the survival of the symbiotic unit in nitrogen deficient tropical waters by providing additional routes of nitrogen uptake and metabolism. The enzymatic pathway of ammonia assimilation from seawater and the re-assimilation of coral ammonium waste by zooxanthellae was studied by examining the affinity of glutamine synthetase for one of its substrates, ammonia. Glutamine synthetase activity was measured in dinoflagellates of the species Symbiodinium microadriaticum found in symbiotic association with various marine coelenterates. Michaelis-Menten kinetics for the substrate ammonia were determined for freshly isolated dinoflagellates from Condylactis gigantea (apparent NH₃ K_m=33 M) and for cultured dinoflagellates from Zoanthus sociatus (apparent NH₃ K_m=60 M). On the basis of the low apparent K_ms for NH₃, it appears that ammonia assimilation by these symbiotic dinoflagellates occurs via the glutamine synthetase/glutamate synthase pathway. Additionally, the uptake of exogenous ammonium by an intact coelenterate-dinoflagellate symbiosis was strongly inhibited by 0.5 mM methionine sulfoximine, and inhibitor of glutamine synthetase.     

Genetic differentiation and reproductive incompatibility among Baja California populations of the copepod Tigriopus californicus

Populations of the harpacticoid copepod Tigriopus californicus were sampled from five sites from San Diego, California, to Playa Altamira, Baja California, Mexico. Allozyme analyses revealed that all the study populations are sharply differentiated genetically. At the extreme, two populations, Punta Baja and Playa Altamira, have no alleles in common at the seven allozyme loci studied. All pairwise interpopulation crosses successfully produced F₂ hybrids except those involving the Playa Altamira population. All crosses using Playa Altamira females failed to produce F₁ hybrids, while Playa Altamira males successfully produced F₁ progeny with females from all other sites. These F₁ offspring, however, were completely sterile (with San Diego and Punta Banda females) or only occasionally produced F₂ offspring (with Punta Morro and Punta Baja females). These results suggest that allopatric differentiation among Baja populations has resulted in exceptionally high levels of genetic divergence and nearly complete reproductive isolation of the Playa Altamira population, which should now be recognized at the semispecies (or perhaps sibling species) level.     

Niche segregation and habitat specialisation of harpacticoid copepods in a tropical seagrass bed

Several harpacticoid copepod species are adapted to an epiphytic lifestyle. Previous studies on tropical seagrass meiofauna mainly focussed on the epiphytic communities and neglected the benthic component. The present study aims to document the benthic harpacticoid copepod communities sampled from different sediment depth horizons adjacent to five seagrass species in the intertidal and subtidal zone of a tropical seagrass bed (Gazi Bay, Kenya). Two benthic copepod communities could be identified mainly based on the tidal position of the samples: a first community was collected near the intertidal seagrasses Halophila ovalis and Halodule wrightii; a second community occurred near the subtidal seagrasses Thalassia hemprichii, Syringodium isoetifolium and Halophila stipulacea. The first community was mainly determined by sediment characteristics (e.g. skewness), while the second community was split off based on organic matter content (% TOM), nutrient and pigment values. A subtle combination of horizontal and vertical niche segregation was reported for the dominant copepod families. Species of the families Thalestridae, Laophontidae and Diosaccidae were structured by tidal position and showed a strong preference for the subtidal zone. The opposite strategy, i.e. a clear preference for the intertidal zone, was found for copepods belonging to the families Paramesochridae and Canuellidae. In addition, Apodopsyllus africanus (Paramesochridae) was well-adapted to stress and was concentrated in the deeper sediment layers near the subtidal seagrasses. On the other hand, Canuellidae, as filter feeders, were concentrated in the upper centimetres of the sediment. The families Ectinosomatidae and Cletodidae did not show any vertical or horizontal segregation. On the species level, however, clear horizontal niche segregation was detected for the family Cletodidae. In addition to the reported ecological results, the study material was used to evaluate different niche definitions. We found tidal position to be the most important factor forcing harpacticoids to specialise. Sediment depth horizon was less powerful in dividing the families into different guilds (from specialists to generalists) based on standardised niche breadth. The present study documents the subtle habitat partitioning of co-existing species in a limited area and its role in sustaining high biodiversity in the community.